Antibiotic derivatives of kanamycin

ABSTRACT

DERIVATIVES OF KANAMYCIN A AND B HAVE BEEN PREPARED WHICH POSSESS SUBSTANTIALLY IMPROVED ANTIBACTERIAL ACTIVITY. AN EXAMPLE OF SUCH AN AGENT IS 1-(L-(-) $-AMINO-AHYDROXYBUTYRYL)-KANAMYCIN A (IVA, BB-K8).

United States Patent ice 3,781,268 ANTIBIOTIC DERIVATIVES 0F KANAMYCINHiroshi KaWaguchL'Takayuki Naito, and Snsumu Nakagawa, Tokyo, Japan,assignors to Bristol-Myers Company, New York, NY.

No Drawing. Continuation-impart of abandoned application Ser. No.162,315, July 13, 1971. This application Jan. 27, 1972, Ser. No. 221,378

Int. Cl. C07c 47/18 US. Cl. 260-210 AB 13 Claims ABSTRACT OF THEDISCLOSURE Derivatives of kanamycin A and B have been prepared whichpossess substantially improved antibacterial activity. An example ofsuch an agent is 1-[L-(-)-'y-amino-ahydroxybutyryl]-kanamycin A [IVa,BB-K8].

CROSS-REFERENCE TO RELATED APPLICATIONS This application is acontinuation-in-part of a copending application, Ser. No. 162,315, filedJuly 13, 1971, and now abandoned.

BACKGROUND OF THE INVENTION (1) Field of the invention This inventionrelates to a semisynthetic l-substituted derivatives of kanamycin A orB, said compounds being prepared by acylating the l-amino-function ofkanamycin A or B with a 'y-amino-u-hydroxybutyryl moiety.

(2) Description of the prior art The kanamycins are known antibioticsdescribed in Merck Index, 8th ed., pp. 597-598. Kanamycin A is acompound having the formula no GHQ-M2 Kanamycin B is a compound havingthe formula en m 3,781,268 Patented Dec. 25, 1973 SUMMARY OF THEINVENTION The compound having the formula Nib-R in which R is OH or NH,,and R is L-(-)-'y-amino-ahydroxybutyryl; or a nontoxic pharmaceuticallyacceptable acid addition salt thcreof is a valuable antibacterial agent.

This invention relates to semi-synthetic derivatives of kanamycin A andB, said compounds being known as 1-[L-(-)-'-amino-a-hydroxybutyryl]-kanamycin A and B and having the formula inwhich R is OH or NH, and R is L-(-)-'y-amino-ahydroxybutyryl; or anontoxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceuticallyacceptable acid addition salt shall mean a mono-, di-, trior tetrasaltformed by the interaction of 1 molecule of compound IV with 14 moles ofa nontoxic, pharmaceutically acceptable acid. Included among these acidsare acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric,hydrobromic, ascorbic, malic and citric acid, and those other acidscommonly used to make salts of amine containing pharmaceuticals.

The compounds of the p'resent invention tem e d (1) Kanamycln ASucclnimide [6'-monobenzyloxycarbonylkanamycin A or B] (2) Compound IIN-hydroxysuccinimlde ester of L-()-lbenzyloxycarbonylamino-c-hydroxybutyrlc acid (3) Compound III Hlrdlcno e e ao-a on c

A preferred embodiment of the present invention is the compound havingthe formula in which R is R is H or L-()-'y-amino-a-hydroxybutyryl and Ris OH or NH wherein R or R must be other than H; or a nontoxicpharmaceutically acceptable acid addition salt thereof.

Another preferred embodiment is the compound of Formula V in which R is0 CoHsCHzO-HJ- R is H and R is OH or NH A more preferred embodiment isthe compound of Formula V in which R is L-()- -amino-whydroxybutyryl, Ris hydrogen and R is OH or NH- A most preferred embodiment is thecompound of Formula V wherein R is H, R is L-(--)-'y-amino-a-hydroxybutyl and R is OH; nontoxic pharmaceutically acceptable acid additionsalt thereof.

Another most preferred embodiment is the compound of Formula V wherein Ris H, H is L-()-'y-amino-uhydroxybutyryl and R is NH or a nontoxicpharmaceutically acceptable and addition salt thereof.

Other most preferred embodiments are the sulfate, hydrochloride,acetate, maleate, citrate, ascorbate, nitrate or phosphate salts ofcompound V.

Another most preferred embodiment is the monosulfate salt of compound V.

Still another preferred embodiment is the disulfate salt of compound V.

The objectives of the present invention have been achieved, by theprovision according to the present invention of the process for thepreparation of the compound having the formula in which R isL-()-'y-amino-a-hydroxybutyryl and R is OH or NIL-I or a nontoxicpharmaceutically acceptable acid addition salt thereof; which processcomprises the consecutive steps of (A) Acylating kanamycin A orkanamycin B with an acylating agent selected from the compounds havingthe formulas omains}:

R4 CH:

R CH3 CH; N 03 O Q X, walls,

0 XCHz&-OH (or a carbudlimide thereof) or O HPCH CH -O 0 11 (or acarbodiimide thereof) in which R and R are alike or different and eachis H, F, Cl, Br, N0 OH, (lower)alkyl or (lower)alkoxy, X is chloro,bromo or iodo, or a functional equivalent as an acylating agent; in aratio of 1 mole or less of acylating agent per mole of kanamycin A or Bin a solvent, preferably selected from the group comprised ofdimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane,1,2-dimethoxyethane, methanol, ethanol, water, acetone, pyridine,N-(lower)alkyl-piperidine, or mixtures thereof, but preferablydimethylformamide, at a temperature below 50 C. and preferably below 25C., to produce the compound having the formula 6 in which Y is a radicalof the formula R 0 CH; 0

CH, L mire-04L R5 (1H3 in which R R and R are as defined above;

(B) Acylating compound II with an acylating agent having the formula OH0 w-NH-Cm-om-oH- -M (VII) in which W is a radical selected from thegroup comprising R4 0 cm 0 ental +0-11 a cm 0 0 Q x-cm-ii, or ii-om-om-bbut preferably M is a radical selected from the group comprising o@ m,LOLA but preferably in which R and R are as above; in a ratio of atleast 0.5 mole of compound VII per mole of compound II, but preferablyin a ratio of about 0.5 to about 1.4, and most preferably in a ratio ofabout 0.8 to about 1.1, in a solvent preferably selected from the groupcomprising a mixture of water and ethyleneglycol dimethyl ether,dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran,

7 propyleneglycol dimethyl ether, or the like but preferably 1:1water-ethylene glycol dimethyl ether, to produce a compound of theformula Pia-NIL! in which R Y and W are as above; and

(C) Removing the blocking groups W and Y from compound III by methodscommonly known in the art, and preferably when W and Y are radicals ofthe formula Q-cm-O-E- by hydrogenating compound III with hydrogen in thepresence of a metal catalyst, preferably selected from the groupcomprising palladium, platinum, Raney nickel, rhodium, ruthenium andnickel, but preferably palladium, and most preferably palladium oncharcoal, in a waterwater miscible solvent system, preferably selectedfrom the group comprising water and dioxane, tetrahydrofuran,ethyleneglycol dimethyl ether, propyleneglycol dirnethyl ether, or thelike, but preferably 1:1 water-dioxane, and preferably in the presenceof a catalytic amount of glacial acetic acid to produce the compound ofFormula IV.

It should be apparent to those knowledgeable in the art that otheragents can be used in the process above to acylate the amine functionsof the intermediate compounds of the instant invention. This disclosureis meant to include all such acylating agents that produce labile amineblocking groups, said labile blocking groups commonly employed in thesynthesis of peptides. The labile blocking groups must be readilyremovable by methods commonly known in the art. Examples of said labileblocking groups and their removal can be found in the review of A.Kapoor, J. Pharm. Sciences, 59, pp. 1-27 (1970). Functional equivalentsas acylating agent for primary amine groups would include correspondingcarboxylic chlorides, bromides, acid anhydrides, including mixedanhydrides and particularly the mixed anhydrides prepared from strongeracids such as the lower aliphatic monoesters of carbonic acid, of alkyland aryl sulfonic acids and of more hindered acids such asdiphenylacetic acid. In addition, an acid azide or an active ester ofthioester (e.g., with p-nitrophenol, 2,4-dinitrophenol, thiophenol,thioacetic acid) may be used or the free acid itself may be coupled withthe kanamycin derivative (II) after first reacting said free acid withN,N-dimethylchloroforminium chloride [cf. Great Britain 1,008,170 andNovak and Weichet, Experientia, XXI/ 6, 360 (1965)] or by the use ofenzymes or of an N,N-carbonyldiimidazole or an N,N'-carbony1ditriazole[cf. Sheehan and Hess, J. Amer. Chem. Soc., 77, 1067 1955)] or ofalkynylamine reagent [cf. R. Buijile and H. G. Viehe, Angew. Chem.,International Edition 3, 582 (1964)], or of a ketenimine reagent [cf. C.L. Stevenes and M. E. Monk, J. Amer. Chem. Soc., 80, 4065 1958)] or ofan isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson and H.Mayer, J. Amer. Chem; Soc., 83, 1010 (1961)]. Another equivalent of theacid chloride is a corresponding azolide, i.e., an amide of thecorresponding acid whose amide nitrogen is a member of a quasiaromaticfive membered ring containing at least two nitrogen atoms, i.e.,imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole andtheir substituted derivatives. As an example of the general method forthe preparation of an azolide, N,N'-carbonyldiimidazole is reacted witha carboxylic acid in equimolar proportions at room temperature intetrahydrofuran, chloroform, dimethylformamide or a similar inertsolvent to form the carboxylic acid imidazolide in practicallyquantitative yield with liberation of carbon dioxide and one mole ofimidazole. Dicarboxylic acids yield diimidazolides. The by-product,midazole, precipitates and may be separated and the imidazolideisolated, but this is not essential. These reaction are well-known inthe art (cf. US. Pat. Nos. 3,079,314, 3,117,126 and 3,129,224 andBritish Pat. Nos. 932,644, 957,570 and 959,054).

Compound Na, 1- [L- -amino-ahydroxybutyryl]- kanamycin A, possessesexcellent antibacterial activity that appears superior to kanamycin Aitself. Illustrated below are two tables showing the minimal inhibitoryconcentrations (MICs) of kanamycin A and compound IVa (BB-K8) against avariety of Gram-positive and Gramnegative bacteria as obtained by theSteers agar-dilution method (Table 1) and the two-fold dilution method(Table 2). Mueller-Hinton Agar Medium was used in the study of Table 1and Heart Infusion Broth was used in the study of Table 2.

TABLE 1 MIC, mg./ml.

Compound Kanamycin A IVa [BB-K8], Organism (6-8198) lot No. 4

1 Alk. jaecalis 11-9423 16 a 2 Alk. faecalz's 11-20648 125 125 3... Eat.cloacae 14-9656... 4 4 4- Eat. species 11-20364. 125 2 5--- Eat. hafm'ae1 11-2067 1 1 6.-. E 001111-0636 2 1 7 E. coli 11-20664. 16 4 8--- E.coli 11-20665 125 1 9 E. coli 11-20507- 32 2 10- E. coli A-20520- 125 411 E. coli 11-20365. 125 1 12-. E. colt A-206 2 2 13.. E coli A20682--125 2 14.- E coli A- 125 3 15. E coli 11 20681 125 2 16. E. coli11-15119 4 4 17. K. pneumoniae 11-9867 4 4 18-- K. species 11-20328.-.125 2 19. K. species 11-20330. 32 32 20-- K. species A20634 125 4 21--K. pneumoniae A-20680.-- 125 4 22- K. pneumoniae A9977.. 1 1 23. Pr.mirabilis A9900-. 2 2 24-- Pr. morga'nii 14-15153 2 2 25- Pr. valgarz's11-9555 2 1 26-- Pr. rettgeri 1143636.... 0. 25 0. 25 27-- P1. mirabih's11-20645 4 4 28. Pr. mirabilis 11-20454. 2 2 29- Pmm'dencia stuartii.A-20615 2 1 30- Providencia alkali jactens A-20676 1 1 31... Pa.aeruginosa A-20229 32 2 32-- Pa. aeruginosa A-9843A-.. 125 16 33-. Pa.aerugtnosa 11-20653. 125 32 34. Pa. species A20601 125, 63 16 35- Ps.species A20621 125 125 36.. Pa. maltophilia 15-20620. 32 1 125 37- Sal.cnteritidis 14-9531 1 0. 5 38.- Sal. derby A-20087 125 1 39 Ser.marcesczns 11-20019. 2 4 40- Ser. marcescens A9933---. 4 8 41. Ser.marcescens 11-20460..- 125 42 42-- Ser. marcescens A20459. 4 16 43-.Ship. flezneri A9684.... 4 4 44.- Aeromonas sp. A-20670 '2 2 45. Arizonasp. 11-20671 2 1 46-. Citrobacter sp. 4-20673 4 4 47. Edwardsiella sp.A.20678 4 4 48-- Staph. aureus .A9606-.'..--- l 1 49. Staph.aureasA-4749. 0. 5 1 50.- Staph. aureus 11-9537. 2 1 51- Staph. aureusA-20610 125 2 52-- Staph. aureus 11-20240 125 8 53-.- Staph. aureusA15197-. 1 2

Mueller-Hinton Medium 1% sheep blood 54. Str. faecalis A-9854- 63 6355-- Str. faecalis 11-9575. 125 125 56. Str. pyogenes 11-20 32, 16 3257-. Str. pyogzmes A-9604 125 125 58-. Str. pyogeaes A15040.-.. 125 12559-- Str. pycgenea 11-20065... 125 125 60-- D. pneumoniae 11-9585 63, 3263 61-- D. pneumoniae A-20159 125 TABLE 2 mg. per day in divided dosesthree or four times a day. MIC m m} Generally the compounds areeffective when administered at a dosage of about 5.0 to 7.5 mg./kg. ofbody weight compo upg every 12 hours. I KanamycinA [BB-K8] 5 CompoundIVb, 1-[L-(-)-'y-amino-a-hydroxybutyryl]- Orgamsm -8196) lot No. 4kanamycin B, possesses excellent antibacterial activity that Dpmumomae+5% Semm 63 63 appears supenor to kanamycin A. Illustrated below is g----l z s 'um keec iu. 1 22 2 Table 4 showing the minimal inhibitoryconcentrations a s Stag Wm M3497 5 M (MICs) of kanamycin A and compoundIVb (BB-K26) 5-.- Staph. aureus 1120239-.-- 125 4 against a variety ofGram-positive and Gram-negative 6..- St h. we 11-20240" 125 4 gg; 532 22 bactena. The MICs are obtained by the Steers agar-d1lu- 8... Enter.species A-20364.. 1 5 2 tion meth 3 6 gp f gg fifi ifig lil g i od(Table 4) on nutrient agar med1um.

C0 1 11-- E. coli 11-12 ML1630 11-20363- 125 2 TABLE 4 12.- E. coli K-1211-9632 2 1 13.. E. coli 11-20664 32 8 MIC (mgJmL) 14-. E. coli 11-20665125 8 15- Pr. mirabilis 11 99000-. 2 16 Bristol Resist; Kaua- 16 P1.morgam'i 11-15153--. 4 16 rganism Code No. to- BBK-26 myciuA 17. Pr.vulgaris A9436 1 2 18.. Pa. ueruginosa 11-20227- 4 1 N J 0.4 0.8 19..P8. species A20499 ea 4 6011111111 1.6 0.8 20.. Fe. aeruginosa A-20653-125 4 20 1.6 1.6 21-- Ps. species 11-20621 125 125 m 0.8 50 22-. Ser.marcescens 11-20019 2 4 0. 8 0. 8 23.- Ser. marcescens 11-20141 16 160.4 50 E colzK-12 0.8 0.4 o.-. 0.8 3.1 The above MIC data showthatcompound IVa (BB- Dom A20665 0.8 50 K8) is superior to kanamycin Ain activ1ty, particularly 25 A967; 8-: g-g against kanamycin A resistantorganisms. S.maryesqens-.].). 1120019 1.6 1.6 The MIC data alsocorrelate well with the m WV? 16- 133 D113 5% 3- 3 sults for all threeorgamsms agamst which kanamycm A Do 299213 1.6 25 and compound IVa weretested. 2 2:; 2 Compound Na and kanamycin A were equally efiec- 3 11 1.612.5 tive in infections of mice caused by kanamycin A-sensitive M0516g1; gg strains of E. coli A15119 and Staph. aureus A9537. Al- Do 3325 2g50 though the CD values (curative dose in 50% of mice 'igggg'gjjjjjjjjjjjj M0355 &3 lethally infected) for Staph. aureus A9537suggest that DO 420358 1 compound IVa is slightly less active thankanamycin A; 6'3 3? this small difference is probably not significantbecause 3.1 12.5 0.3 50 the dose levels were far apart (5 -d1lut1ons) 5o50 Against the kanamycin-resistant strain of E. coli 0 Do 0.8 0.8A20520, kanamycin A as expected, was not effective in p mz L6 vivo,whereas compound IVa demonstrated a marked 40 PT morgam l-g lgprotective action. Compound IVa was approximately 10 1 times more activeagainst this E. coli strain when adminwilt/m aureus 20 1. 6 0. 8 isteredin a 4-treatment reglmen rather than 1n a 2-treats aureus Smlth 0.8 0.4.S. aureus 20919.- 1. 6 0. 8 ment one. S 1 2 1)?) h; 3 u ea 1 T F M.flaws 0.4 0.8 A comparison of in vitro and m v1vo activities of CompoundNa and B. subtilis POI-219 0. 1 0. 1 Kanamycin A St. p 23 25 25 Dic 1.61.6 Staphz Zoccus ESChETZChllZ Escherichia St. pyogenea Digonnet A960412. 5 25 aureus A9537 coli A15119 coli A20520 Do A20065 25 25 Test D. p25 Compounds No. MIC CDs MIC CD50 MIC CD50 50 M, tum 607 6.3 0.8Mycobacterium 607 D105 KM 100 100 Compound IV 1 1 2 0X2 2 2X2 2 66 2Mycobacterium 607 D107..- KM 100 100 2 5X Mycobacterium phlei 1. 6 1. 6KanamyoinA 1 2 0 5X2 4 4X2 125 200X2 M tum rrmar 6.3 1.6 2 200X4Mycobacterium H3 Rv 0. 8 0. 4

MIC=minimum inhibitory concentration gJmL). Tests conducted Determinedby tube dilution method. as described by Chrisholm et al,. (Antimicrob.agents and Chemother- Norm-KM is kanamycin A; GM is gentamycin.

spy-d969, p. 244. 1970) using Mueller-Hinten agar as the test medium.

CD 0=Cnrative dose, 50% (mg./kg./treated times number of treatments).Mice were treated subcutaneously at 1 and 4 hours pest-infection when 2treatments were administered and at 0, 2, 4, and 6 hours postinfectionwhen 4 treatments were given. Other aspects of the test were carried outas described by Price et al. (I, of antibiotics 22:1. 1969).

=Not tested,

The compounds IV are valuable as antibacterial agents, nutritionalsupplements in animal feeds, therapeutic agents in poultry and animals,including man, and are especially valuable in the treatment ofinfectious diseases caused by Gram-positive and Gram-negative bacteria.

The compounds IV when administered orally are useful as an adjunctivetreatment for preoperative sterilization of the bowel. Both aerobic andanaerobic flora which are suseptible to these drugs are reduced in thelarge intestine. When accompanied by adequate mechanical cleansing, theyare useful in preparing for colonic surgery.

The compounds IV are effective in the treatment of systemic bacterialinfections when administered parenterally in the dosage range of about250 mg. to about 3000 Code No. Kanamycln sensitivity E. coli .l'uhlA15119 Sensitive. E. colt ML-1630 A20363 Resistant (phosphorylation).Ps. aerugmosa D15 Moderately resistant. .Ps. aeruginosa D113 Highlyresistant (phosphorylatlon).

Compound IVb showed slightly better activity against equally effectiveagainst E. coli Juhl A15119 at a dose of 6.3 mg./kg. in mice. CompoundIVb was most effective against kanamycin B resistant E. coli ML-1630 ata dose of 6.3 mg./kg. vs. -400 mgJkg. for kanamycin B.

1 1 Compound IBb showed slightly better activity against moderatelyresistant Ps. aeruginosa D15 than kanamycin B. Compound IVb showedsubstantially better activity against highly resistant Ps. aeruginosaD-113 than kanamycin B (see Table 5 below).

TABLE 5 E. coli Juhl A15119 E. coli ML-1630 Dose (s.e.), mgjkg. BB-K26KM-B BB-KZG KM-B Pa. aeruqinosa D15 P8. aeruginosa. D-113 Dose (5.0.),Doses (s.c.),

mgJkg. BB-K26 KM-B mgJkg. BB-K26 KM-B NOTE.KMB is kanamycln B. Thefigures shown as 0/5 1/5, 5/5, etc,. indicate the number of survivinganimals per five animals challenged; for example, 5/5 indicates 5 of 5animals surviving the lethal dose of challenge organism when treatedwith kanamycin B or BB-K26.

The compounds of the present invention are the subject matter of ourco-pending US. patent application, Ser. No. 162,315, filed July 13,1971. Subsequent to filing the parent application, it was determinedthat the wrong structures were assigned to the compounds of the presentinvention. It was originally thought the L-()-'y-amino-ahydroxybutrylmoiety was on the 3" position of kanamycin A and B, when in fact theprocess of production, as illustrated by the examples, placed theL-()-'y-amino-uhydroxybutyryl moiety on the 1-position. While thecompounds claimed herein were in fact produced by the process of theparent application and are described therein, those compounds weremistakenly identified as to their structure in the parent application.

EXAMPLES Example 1 Preparation ofL-(-)-'y-benzyloxycarbonylamino-a-hydroxybutyric acid(VI).L-(--)-'y-amino-a-hydroxybutyric acid (7.4 g., 0.062 mole was addedto a solution of 5.2 g. (0.13 mole) of sodium hydroxide in 50 ml. ofwater. To the stirred solution was added dropwise at 0-5 C. over aperiod of 0.5 hour, 11.7 g. (0.068 mole) of carbobenzoxy chloride andthe mixture was continued to stir for one hour at the same temperature.The reaction mixture was washed with 50 ml. of ether, adjusted to pH 2with dilute hydrochloric acid and extracted with four 80-ml. portions ofether. The ethereal extracts were combined, washed with a small amountof saturated sodium chloride solution, dried with anhydrous sodiumsulfate and filtered. The filtrate was evaporated in vacuo and theresulting residue was crystallized from benzene to give 11.6 g. (74%) ofcolorless plates; melting point 78.5- 79.5 C., [0c] =4.5 (c.=2, CHgOH).Infrared (IR) [KBr]: IR (KBr) 'y 1740, 1690 cmr Nuclear magneticresonance (NMR) (acetone-d 6 (in p.p.m. from TMS) 2.0 (2H, m.), 3.29(2H, d-d, J-=6.7 and 12 Hz.), 4.16 (1H, d-d, 1:45 and 8 Hz.), 4.99 (2H,s.), 6.2 (2H, broad), 7.21 (SH, s.).

Analysis.-Calcd. for C I-I NO (percent): C, 56.91; H, 5.97; N, 5.53.Found (percent): C, 56.66; H, 5.97; N, 5.47.

'Example 2 N-hydroxysuccinimide ester ofL-(-)-'y-benzy1oxycarbonylamino-u-hydroxybutyric acid (VII).-A solutionof 10.6 g. (0.042 mole) of VI and 4.8 g. (0.042 mole) ofN-hydroxysuccinimide 1 in 200 ml. of ethyl acetate was i G. W. Andersonet 211., J. Am. Chem. Soc., 86, 1839 (1064).

cooled to 0 C. and then 8.6 g. (0.042 mole) of dicyclohexylcarbodiimidewas added. The mixture was kept overnight in a refrigerator. Thedicyclohexylurea which separated was filtered oif and the filtrate wasconcentrated to about 50 ml. under reduced pressure to give colorlesscrystals of VII which were collected by filtration; 6.4 g., M.P.121-1225 C. The filtrate was evaporated to dryness in vacuo and thecrystalline residue was washed with 20 ml. of a benzene-n-hexane mixtureto give an additional amount of VII. The total yield was 13.4 g. (92%[6], 1.5 (c.=2, CHCl IR (KBr) I 1810, 1755, 1740, 1680 cm.- NMR(acetone-d 5 (in p.p.m. from TMS) 2.0 (2H, m.), 2.83 (4H, s.), 3.37 (2H,d-d, J=6.5 and 12.5 Hz.), 4.56 (1H, m.), 4.99 (2H, s.), 6.3 (2H, broad),7.23 (5H, s.).

Analysis.-Calcd. for C H N O (percent): C, 54.85; H, 5.18; N, 8.00.Found (percent): C, 54.79, 54.70; H, 5.21, 5.20; N, 8.14, 8.12.

Example 3 Preparation of 1 [L()-'y-benzyloxycarbonylaminoa-hydroxybutyryl] 6' carbobenzoxykanamycin A(IIIa).A solution of 1.6 g. (4.6 mmoles) of VII in 40 ml. ofethyleneglycol dimethyl ether (DME) was added dropwise to a stirredsolution of 2.6 g. (4.2 mmoles) of 6'-monobenzyloxycanbonylkanamycin A(II) in 40 ml. of 50% aqueous ethyleneglycol dimethyl ether and themixture was stirred overnight. The reaction mixture was evaporated underreduced pressure to give a brown residue of IIIa which was used for thenext reaction without further purification.

Example 4 Preparation of l [L-()-'y-amino-a-hydroxybutyryl]- kanamycin A(IVa).The crude product IIIa from Example 3 was dissolved in 40 ml. of50% aqueous dioxane and a small amount of insoluble material was removedby filtration. To the filtrate was added 0.8 ml. of glacial acetic acidand 1 g. of 10% palladium-on-charcoal and the mixture was hydrogenatedat room temperature for 24 hours in a Parr hydrogenation apparatus. Thereaction mixture was filtered to remove the palladium catalyst and thefiltrate was evaporated to dryness in vacuo. The residue was dissolvedin 30 ml. of water and chromatographed on a column of CG-50 ion exchangeresin (NH type, 50 cm. x 1.8 cm.) The column was washed with 200 ml. ofwater and then eluted with 800 ml. of 0.1 N NH OH, 500 ml. of 0.2 N NH0H and finally 500 m1. of 0.5 N NH OH. Ten-milliliter fractions werecollected and fractions 146 to 154 contained 552 mg. (22%, based oncarbobenzoxy kanamycin A, II) of the product IV which was designatedBB-K8 lot 2. MP. 187 (dec.). Relative potency against B. subtilis (agarplate)=560 meg/mg. (standard: kanamycin A free base).

A solution of 250 mg. of BB-K8 lot 2 in 10 ml. of water was subjected tochromatography on a column of CG-50 (NH type, 30 cm. x 0.9 cm.). Thecolumn was washed with 50 ml. of water and then eluted with 0.2 N NH OH.Ten milliliter fractions were collected. Fractions 50 to 63 werecombined and evaporated to dryness under reduced pressure to give 98 mg.of the pure product, K8 lot 2-1. M.P. 194 (dec.). [a] (c.=2, H O).Relative potency against B. subtilis (agar plate)=960 meg/mg. (standard:kanamycin A free base).

Analysis.Calcd. for C H N O -2H CO (percent): C, 40.62; H, 6.68; N,9.87. Found (percent): C, 40.21, 39.79; H, 6.96, 6.87; N, 9.37, 9.49.

Example 5 Preparation of N (benzyloxycarbonyloxy)succinimide.N-hydroxysuccinimide 2 (23 g., 0.2 mole) was dissolved in asolution of 9 g. (0.22 mole) of sodium hydroxide in 200 ml. of water. Tothe stirred solution was 2 G. W. Anderson et 11.1., J, Am. Chem. Soc,86, 1839 (1964).

added dropwise 34 g. (0.2 mole) of carbobenzoxy chloride withwater-cooling and then the mixture was stirred at room temperatureovernight to separate the carbobenzoxy derivative which was collected byfiltration, washed with water and air-dried. Yield 41.1 g. (82%).Recrystallization from benzene-n-hexane (10: 1) gave colorless prismsmelting at 78-79" C.

Example 6 I Preparation of 6'-carbobenzoxykanamycin A.-A solution of42.5 g. (90 mmoles) of kanamycin A free base in 450 ml. of water and 500ml. of dimethylformamide (DMF) was cooled below C. and stirredvigorously. To the solution was added dropwise over a period of abouttwo hours a solution of 22.4 g. (90 mmoles) of N-(benzyloxycarbonyloxy)succinimide in 500 ml. of DMF. The mixture was stirred at -10 to 0 C.overnight and then at room temperature for one day. The reaction mixturewas evaporated under reduced pressure below about 50 C. The oily residuewas dissolved in a mixture of 500 ml. water and 500 ml. butanol, themixture being filtered to remove insoluble material and separated intotwo layers. The butanol and aqueous layers were treated withbutanol-saturated water (500 ml. 2) and water-saturated butanol (500 ml.2), respectively, using a technique similar to counter currentdistribution. The three aqueous layers were combined and evaporated todryness under reduced pressure to give an oily residue, a part of whichcrystallized on standing at room temperature. To the residue includingthe crystals was added about 100 ml. of methanol, which dissolved theoil and separated it from the crystals. After adding about 300 ml. ofethanol, the mixture was kept at room temperature overnight to give acrystalline mass which was collected by filtration. It weighed 44 g. Theproduct contained a small amount of kanamycin A as indicated by thinlayer chromatography using 11 propanol pyridine acetic acid-water(15:10:3z12) as the solvent system and Ninhydrin as the spray reagent.

The crude product was dissolved in 300 ml. of water and chromatographedon a column (30 mm. diameter) of CG-SO ion-exchange resin (NHJ type, 500ml.). The column was irrigated with 0.1 N ammonium hydroxide solutionand the eluate was collected in 10-ml. fraction. The desired product wascontained in tube numbers 10- 100, while kanamycin A recovered fromslower-moving fractions and the position isomer(s) of the product seemedto be contained in the faster-moving fractions. The fractions 10-110were combined and evaporated to dryness under reduced pressure to give24.6 g. (45%) of a colorless product 6-carbobenzoxykanamycin A (II) [6'-Cbz-kanamycin A], which began to melt and color at 204 C. and decomposedat 212 C. with gas evolution. [OLZID +106 (C.*=2, H)-

Ri value TLC (silica gel F254; Ninhydrin) solvent systemn-PrOH-pyridine-AcOH-JizO Kana- 6-Cbz-Kanamyein A mycin A 2 Minor. 8Detected by anthrone-suliuric acid.

The final product was found to be accompanied by two minor components byTLC with one of the solvent systems tested. However, the final productwas used without further purification for the preparation of BB-K8 (I).

Example 7 Preparation of L-()-'y-amino-a-hydroxybutyric acid fromambutyrosin A or B or mixtures thereof.Ambutyrosin A (5.0 gm.) [U.S.Pat. No. 3,541,078, issued Nov.

17, 1970] was refluxed with 160 ml. of 0.5 N sodium hydroxide for onehour. The hydrolysate was neutralized with 6 N HCl and chromatographedon a column of (36-50 (NH type). The desiredL-(-)-'y-amino-ahydroxybutyric acid was isolated by developing thecolumn with water and removing the water by freeze drying. TheL-()-'y-amino-a-hydroxybutyric acid is characterized as a crystallinematerial having a M.P. of 212.5-214.5 0. [column 2, lines 31-38, US.Pat. No. 3,541,078].

Example 8 Preparation of 6'-carbobenzoxykanamycin B.To a chilledsolution of 8.1 g. (0.0168 mole) of kanamycin 'B in 120 ml. of water andml. of 1,2-dimethoxyethane was added dropwise with stirring a solutionof 4.2 g. (0.0168 mole) of N-(benzyloxycarbonyloxy)succinimide in 40 ml.of 1,2-dimethoxyethaue. The reaction mixture was stirred overnight andevaporated under reduced pressure. The residue was dissolved in ml. ofWater and shaken twice with 5 0 ml. of water-saturated n-butanol. Theaqueous layer was separated and adsorbed on a column of 100 ml. of CG-SO(NH type). The column was washed with 200 ml. of water, eluted with 0.05N NH OH. The eluate was collected in 10-ml. fraction. Fractions 121 to180 were collected, evaporated and freeze-dried to give 1.58 g. (15%) ofthe desired product. Fractions 1 to were evaporated andre-chromatographed on CG-SO (NH to give 1.21 g. (12%) of the product.M.P. 151-152" C. (dec.). [04 +104 (c.=2.5, H O). 7 1710 cm.-

Analysis.Calcd. for C H N O (percent): C, 50.56; H, 7.02; N, 11.34.Found (percent): C, 50.71; H, 7.38; N, 11.48.

TLC (silica gel F254), Rf 0.03 in n-PrOH-AcOH-H O (15:10:3z12); Rf 0.16in acetone-AcOH-H 0 (20:6:74).

Example 9 Preparation of l- [L--'y-benzyloxycarbonylaminoa-hydroxybutyryl] 6' carbobenzoxykanamycin B(IIIb).To a stirred solution of 1.85 g. (3.0 mmoles) of6'-carbobenzoxykanamycin B in 40 ml. of H 0 and 50 ml. of1,2-dimethoxyethane was added all at once at 5 C. 1.1 g. (3.1 mmoles) ofN-(L-y-carbobenzoxyamino-u-hydroxybutyryloxy)succinimide. The reactionmixture was stirred overnight at room temperature and subjected tohydrogenolysis without isolating the carbobenzoxy derivative (IIIb). TLC(silica gel F254), Rf 0.06 (starting material), 0.41, 0.57(n-PrOH-pyridine-AcOH- H O=15:10:3:12), Rf 0.11 (starting material)0.21, 0.34, 0.46 (acetone-AcOH-H O=20:6:74).

Example 10 Preparation of 1-[L-(-)-'y-amino-a-hydroxybutyrynkanamycin B(IVb).-To the solution obtained in Example 9 was added 0.2 g. of 10%palladium on charcoal. After the mixture was hydrogenated underatmospheric pressure for five hours, an additional amount of 10%palladium on charcoal (0.1 g.) and 10 ml. of water were added.Hydrogenation was continued overnight. The reaction mixture wasfiltered, the filtrate was evaporated under reduced pressure,the'residue was dissolved in 50 ml. of water and chromatographed on acolumn of CG- 50 (NH 1.2 cm. X 50 cm.). The column was washed with 200ml. of water and then eluted with 500 ml. of 0.1 N NH OH, 500 ml. of 0.2N NH OH, 900 ml. of 0.5 N NH OH and 500 ml. of 1 N NH4OH. The efl luentswere collected in 10-ml. fraction. Kanamycin B was recovered fromfractions 60 to 76 in 32% recovery (459 mg.). Fractions 128-138 werecollected, evaporated under reduced pressure and freeze-dried to give318 mg. (17%, based on carbobenzoxy kanamycin B) of BB-K26 (IVb) M.P.186-187 C. (dec.). +78 (c.=1.15, H O). 10 0 GEL-1' Analysis.-Calcd. forC H N O -H CO (percent): C, 42.72; H, 7.17; N, 13.00. Found (percent):C, 42.23; H, 7.19; N, 12.37.

TLC (silica gel F254, Ninhydrin), Rf 0.11 in acetone- AcOH-H O(20:6:74); Rf 0.19 in CHCl -MeH-concentrate NH OH-H O (1 :4:2: 1).

Fractions 201 to 222 were combined, evaporated under reduced pressureand freeze-dried to give 209 mg. (12%) of another active component whichwas designated BB- K27. M.P. 183-184 C. (dec.). 73:0 1750 cm.-

Analysis.Calcd. for C H N O -H CO (percent): C, 42.72; H, 7.17; N,13.00. Found (percent): C, 42.25; H, 6.93; N, 12.18.

TLC (silica gel F254), Rf 0.15 in acetone-AcOH-H O (20:6:74); Rf 0.07 inCHCl -MeOH-concentrate NH OH-H O (1:4:2:1).

Example 11 Preparation of L()--, -amino-a-hydroxybutyric acid fromDL-a-hydroxy 7 phthalimidobutyric acid.-(A) DehydroabietylammoniumL-a-hydroxy 7 phthalimidobutyrate: To a solution of 25 g. (0.1 mole) of2-hydroxy- 'y-phthalimidobutyric acid in 200 ml. of ethanol was added asolution of 29 g. (0.1 mole) of dehydroabietylamine in 130 ml. ofethanol. The solution was shaken vigorously for a minute'and stood atroom temperature for five hours during which time fine needlescrystallized out. The crystals were collected by filtration, washed with50 ml. of ethanol and air-dried to obtain 30.1 g. (56%) of adiastereomer of the dehydroabietylamine salt. M.P. 9394 C. +15 (c.=2.5,MeOH). Recrystallization from 300 ml. of ethanol gave 23.2 g. (43%) ofthe pure product. M.P. 94-95 C. +10.8 (c.=2.5, MeOH). Furtherrecrystallization did not change the melting point and the specificrotation.

Analysis.Calcd. for C H N -O 'H O (percent): C, 69.54; H, 8.02; N, 5.07.Found (percent): C, 69.58; H, 8.08; N, 5.07.

(B) L-( -)-'y-amino-a-hydroxybutyric acid: To a solution of 1.5 g.(0.014 mole) of sodium carbonate in 40 ml. of water were added 5.3 g.(0.01 mole) of dehydroabietylammonium-L-a-liydroxy "yphthalimidobutyrate and 60 ml. of ether. The mixture was shakenvigorously until all of the solid had dissolved. The ether layer wasseparated. The aqueous solution was washed twice with 20-ml. portions ofether and evaporated to 15 ml. under reduced pressure. To theconcentrate was added m1. of concentrated hydrochloric acid and themixture was refluxed for ten hours. After cooling, separated phthalicacid was removed by filtration. The filtrate was evaporated underreduced pressure. The residue was dissolved in 10 ml. of water and thesolution was evaporated to dryness. This operation was repeated twice toremove excess hydrochloric acid. The residual syrup was dissolved in 10ml. of water and filtered to remove a small amount of insoluble phthalicacid. The filtrate was adsorbed on a column of IR-120 (H'*', 1 cm. x 35cm.), the column was washed with 300 ml. of water and eluted with 1 Nammonium hydroxide solution. The eluate was collected in -ml. fraction.The Ninhydrin positive fractions 10 to 16 were combined and evaporatedunder reduced pressure to give a syrup which crystallized gradually. Thecrystals were triturated with ethanol, filtered and dried in a vacuumdesiccator to give 0.78 g. (66%) of L-(--)-'yamino-ahydroxybutyric acid.M.P. 206-207" C. [ch -29 (c.=2.5, H O). The IR spectrum was identicalwith the authentic sample which was obtained from ambutyrosin.

Example 12 Preparation of the monosulfate salt of1-[L-(-)-'yamino-a-hydroxybutyryl]kanamycin A or B.-One mole of1-[L-(--)-'y-amino-a-hydroxybutyryl]kanamycin A or I. Saito et 211.,Tetrahedron Letters, 1970, 4863.

B is dissolved in 1 to 3 liters of water. The solution is filtered toremove any undissolved solids. To the chilled and stirred solution isadded one mole of sulfuric acid dissolved in 500 ml. of water. Themixture is allowed to stir for 30 minutes, following which cold ethanolis added to the mixture till precipitation occurs. The solids arecollected by filtration and are determined to be the desired monosulfatesalt.

Example 13 Preparation of the disulfate salt of 1-[L-()-'y-aminoahydroxybutyryl]kanamycin A (BB-K8-2H SO Thirty-five grams of1-[L-()-'y-amino-a-hydroxybutyryl]kanamycin A (as the monobicarbonatetrihydrate) was dissolved in 125 ml. of deionized water. A pH ofapproximately 9.0 was noted. The pH Was lowered to 7-7.5 with 50% MN.sulfuric acid.

Eight and one-half grams of Darco G-60 (activated charcoal) was addedand the mixture was slurried at ambient room temperature for 0.5 hour.The carbon was removed by suitable filtration and washed with 40 ml. ofwater. The water wash was added to the filtrate.

The combined filtrate-wash above was adjusted to pH 2-2.6 with 50% v./v.sulfuric acid. A large amount of carbon dioxide was evolved. Thesolution was left at house vacuum with stirring for 20 minutes to expeladditional carbon dioxide.

Eight and one-half grams of Darco G-60 was added to the degassedsolution. The mixture was slurried for 0.5 hour at ambient roomtemperature. The carbon was removed by suitable filtration and washedwith 35 ml. of deionized water. The water was added to the filtrate.

The combined filtrate-wash was adjusted to pH 1-l.3 with 50% v./v.sulfuric acid. This solution was added with rapid stirring over a 10minute period to 600-800 ml. of methanol (3-4 volumes of methanol). Themixture was stirred for 5 minutes at pH 1-1.3, passed through a meshscreen, stirred for 2 minutes and allowed to settle for 5 minutes. Mostof the supernatant was decanted. The remaining slurry was suitablyfiltered, washed with 200 ml. of methanol and vacuum dried at 50 C. for24 hours. The yield of amorphous BB-K8 (dihydrogen sulfate): was 32-34grams; [0;] H O==+74.75, decomposition at 220230 C.

Elemental analysis (on dry basis) Found Theory Percent O 32. 7, 33. 5,32.3 33. 5 Percent N 8. 78, 8. 7, 8. 2, 8. 8 8. 97 Percent S 8. 2

8. 75, 8. 9, 7. 8, 8. 8 5 Percent ash N11 Karl Fisher water content:2.33, 1.79, 2.87% (theory for monohydrate is 2.25% water). This salt ishygroscopic but not diliqueseent. After storage of an aliquot in air atroom temperature for 18 hours the water content ncreased to 9.55, 9.89%(theory for a pentahydrate is 10.33% water).

We claim: 1. A compound having the formula in which R is 17 R isL-()-'y-amino-a-hydroxybutyryl or L'ybenzyloxycarbonylamino-a-hydroxybutyryl, and R is OH or NH or anontoxic pharmaceutically acceptable acid addition salt thereof.

2. The compound of claim 1 wherein R is R is L 'y benzyloxycarbonylaminoa hydroxybutyryl and R is OH.

3. The compound of claim 1 wherein R is NH or a nontoxicpharmaceutically acceptable acid addition salt thereof.

6. The monosulfate salt of the compound of claim 4.

7. The monosulfate salt of the compound of claim 5.

8. The disulfate salt of the compound of claim 4.

9. The disulfate salt of the compound of claim 5.

10. The mono or polyhydrates of the compound of claim 8.

11. The mono or polyhydrates of the compound of claim 9.

12. The mono or polyhydrates of the compounds of claim 4.

13. The mono or polyhydrates of the compounds of claim 5.

References Cited UNITED STATES PATENTS 3,032,547 5/1962 Rothrock et al.260-210 K 3,652,535 3/1972 Keil et a1. 260-210 AB JOHNNIE R. BROWN,Primary Examiner US. Cl. X.R.

